This invention relates generally to the field of detecting hazardous chemicals. More particularly, the present invention relates to methods, compositions, devices and kits thereof useful in the detection of chemical warfare agents and insecticides.
Organophosphorus and organosulfur (OP refers to both types) compounds, are used extensively in insecticides and are highly toxic to many organisms including humans. Insecticide residues are found in soil and groundwater, and the detection of these residues is important for their elimination from the environment and to protect the health of both humans and animals. OP compounds are also used in nerve agents, such as sarin, phosphine, soman, and tabun, for chemical warfare purposes.
These agents are some of the most potent toxic agents and are specific inhibitors of acetylcholinesterase (AChE). The sequel to AChE poisoning is a cholinergic crisis in man; the clinical effects are directly related to acetylcholine accumulation. Nerve agents are classified into G agents (GD, soman; GB, sarin; and GA, tabun) and the V agents (VX). These agents differ in physical properties, for example, VX has a much lower vapor pressure than the G agents. However, the toxicity and main effects of the agents are very similarxe2x80x94inhibition of acetylcholinesterase and subsequent breakdown of the normal operation of the automatic and central nervous systems. Thus, detection of organophosphorous compounds is of paramount importance to prevent casualties due to OP exposure.
The need for the reliable determination of these cholinesterase inhibitors has led to the development of a number of sophisticated instrumental methods, mostly involving the use of gas and liquid chromatography and mass spectrometry. Also a number of liquid phase chemiluminescence procedures have been developed for the determination of inorganic and organic species mostly utilizing the luminol and peroxyoxalate reactions. See Robards K. and Worsfold P. J., (1992) Anal. Chem. Acta, 266:147.
These traditional methods are not practical for individual use as the methods are time consuming and complicated and the instruments utilized are expensive, non-portable and require high maintenance. Additionally, the measurement of nerve agents in mixtures with these traditional methods requires cumbersome extraction and manipulation procedures.
Thus, biosensors were developed as an alternative to the traditional gas and liquid chromatography and mass spectrometry technology. Generally, biosensors include those which are enzyme-based and bioaffinity-based. An enzymatic biosensor uses an enzymatic or metabolic process to detect a reaction product which occurs between an incoming substrate and an immobilized enzyme. A bioaffinity sensor relies on a biological binding event of a target substance.
The prior art biosensors are electronic devices which produce electronic signals as the result of biological interactions. These biosensors comprise a biological receptor linked to an electronic transducer in such a way that biochemical activity is converted into electrical activity. The electronic component of the biosensors measure voltage, amperage, wavelengths, temperature, or mass. See Lowe, C. R. (1984) Biosensors 1:3-16.
Biosensors are widely used to detect biological, pharmacological, or clinically important compounds. Generally, enzyme biosensors are selective, sensitive and specific. They are portable, simple and easy to use. Enzymatic biosensors can detect only those substances of interest and ignore all other environmental and biological interference.
Various cholinesterases (ChEs) biosensors have been described. These biosensors comprise ChEs non-covalently immobilized on a support. Cholinesterases have been immobilized on a full gamut of solid and gel supports such as glass, silica, ion-exchange resins, agarose, and nylon supports. Ideally, the preferred methods of immobilizing enzymes on solid supports have high coupling rates and the preferred biosensors retain enzymatic activity and maintain stability. However, biosensors which have non-covalently bound enzymes possess undesirable characteristics such as enzymatic instability at ambient and/or denaturing conditions, a propensity of the enzymes to leach from the surface to which it was non-covalently bound, and a short half-life in solution.
Generally, most methods which covalently bind enzymes to polymers utilize harsh or protein unfriendly conditions diminish enzymatic activity and stability. Although U.S. Pat. No. 4,342,834 discloses a method of making isocyanate-based polyurethane foams wherein enzymes having varying degrees of activity are covalently linked, it does not disclose a material useful for the detection of OP compounds nor does it disclose a method of using the material for the detection of OP compounds.
Furthermore, it does not disclose a method of immobilizing enzymes on a porous support, whereby air induced shear forces are reduced which results in the retention of enzymatic activity.
In one embodiment the invention relates to a material comprising a porous support wherein an enzyme is immobilized upon or within which material is suitable for use as a biosensor for the detection and measurement of hazardous compounds such as OP compounds.
In a preferred embodiment, the material comprises an enzyme that is covalently bound to a porous support, the immobilized enzyme exhibits enzymatic stability at extreme temperatures and/or denaturing conditions, and similar kinetic characteristics of the soluble form. The enzyme does not leach from the porous support and the material retains enzymatic activity after prolonged storage.
In another embodiment, a plurality of materials comprising a porous support wherein a plurality of enzymes is immobilized upon or within which the plurality of materials is suitable for use as a differential biosensor having multiple zones for detecting the presence of specific OP compounds. In a preferred embodiment, the dimensions of the biosensor are in the range from about 0.25xe2x80x3xc3x970.25xe2x80x3xc3x970.03125xe2x80x3 to about 6xe2x80x3xc3x974xe2x80x3xc3x970.25xe2x80x3.
Yet another embodiment of the invention relates to methods of making a material comprising a porous support wherein a plurality of enzymes is immobilized which material is suitable for use as a biosensor for detecting hazardous compounds such as OP compounds. Synthesis of the porous support involves the use of a prepolymer such as polyether and a surfactant such as P-65. In a preferred embodiment, the method of making the material reduces air induced shear forces or shear stress which reduce enzymatic activity. During synthesis of the material by prior art methods, for example a mixing drill, the enzymes utilized are subjected to fluid forces or shear stress. Use of a device that gently folds the components into one another greatly reduces these fluid forces or shear stress, and is the preferred device for enzymes, specifically enzymes that are sensitive to the high shear forces of the drill mixing device. The low shear mixing device more than doubles the resultant AChE or BChE immobilized enzyme activity when compared to an identical mixture prepared with the high shear device. Additionally, use of additives such as surface-acting polymers, e.g. P-65, or low concentrations of glycerol protects against enzyme denaturation induced by shear forces.
As disclosed herein, one of ordinary skill in the art may synthesize a variety of porous supports with the various prepolymers and surfactants available.
When immobilized, the enzymes of the biosensors are stable under extreme temperatures and/or denaturing conditions. These enzymes include cholinesterases, choline oxidase, hydrogen peroxidase, organophosphate hydrolase, phosphotriesterase, laccase, and derivatives thereof.
The biosensors are suitable for detecting and quantifying OP chemicals in gases and liquids and on solids. For example, the biosensor may be used to detect OP compounds in water and/or soil. The biosensor may be used to detect OP compounds on natural, synthetic and/or biological surfaces, such as wood, plastic and skin. By using mammalian cholinesterases such as FBS-AChE or Eq-BChE rather than Eel Cholinesterase as is found in the M272 ticket (currently used to detect organophosphate compounds; available from Truetech, Inc.), the immobilized sensor will display the same sensitivity to OPs that mankind is suseptible to now, or any new or novel OPs that might be produced in the future against mankind.
In one embodiment, the biosensor comprises at least one indicator to indicate the presence of an OP compound by fluorescent, chromogenic or chemiluminescent determination.
In another embodiment the biosensor comprises a carbon electrode for the detection of an OP compound by determining cholinesterase activity.
In another embodiment, the amount and source of the various OP compounds may be determined.
Yet another embodiment relates to kits. Kits may include, along with the biosensor, the indicators for both quantitative or qualitative detection of OP compounds and means for transmitting results to a central collection point, e.g. computer, satellite uplinks, radio relays, handheld battery operated measuring devices, etc. For example, one may quantitatively analyze the OP compounds by using a handheld battery operated measuring devices and interfacing with a computer to calculate reaction rates which rates may be relayed to a central collection point. These kits may also contain instructions, solutions and compositions needed for operation. The compositions and solutions may be placed in containers, test tubes, etc.
Other embodiments include the methods of using the instant biosensors for the quantitative or qualitative determination of hazardous compounds such as OP compounds.